Experimental Study On Tissue-engineered Bone For Repair Bone Defects

Large bone defects are commonly seen in orthopaedic clinical work, but effective therapeutic methods for bone regeneration are still not available. The technique of tissue engineering provides a new way in repairing bone defects. There are many advantages: (1) the grafts possess biological activity, (2) the source of material is extensive, (3) the shape of grafts can be changed for adaptation, (4) the antigenicity isn't obvious if the seeded cells are autograft, (5) it avoids the surgical complications caused by obtaining bone from the host. American scientist Vacanti obtained calf tissue-engineered artificial bone with certain morphology, structure and function by bioengineering technology in 1994. Nowadays, many experiments have been designed to solve initial problems, such as optimal accumulation of seeded cells, preparation of biodegradable scaffold composites, action of growth factors, construction and culture of cell-scaffold complex, efficiency of the tissue-engineering bone in repairing bone defects. Experiments of repair and functional recovery of segmental bone defects with tissue-engineered bone have been reported and this technique has been used in clinical trial in a few country, but this is not good enough, it needs to be improved. It predicts that the times of ' from organ transplant to organ copy ' is coming as the research of tissue engineering has been developed quickly.The ultimate goal of tissue engineering is to translate basic information into suitable clinical protocols, but many important problems still need to be explored for clinical treatment. This study was carried out for essential techniques, mesenchymal stem cells (MSCs) as the seeded cells, fluorescent protein as tracer, fibrin gel as novel material for construction cell-scaffold complex. Together our preliminary experiments prepare to find out the viability and migration of allogeneic MSCs into subcutaneous tissue and construct a new vector containing human hypoxia-inducible factor-1α gene for angiogenesis.Contents:1. Expanded culture of human MSCs in vitro under optimal conditions and choice reliable methods of osteogenic differentiation.2. Labeling MSCs with enhanced green fluorescent protein (EGFP) or red fluorescent protein (RFP) and then investigation diversification of the biological characteristics.3. Explored a novel technique for construction cell-scaffold complex with fibrin gel.4. Transplantation of allogeneic mesenchymal stem cells into rabbit subcutaneous tissue.5. Construction and identification of human hypoxia-inducible factor-1 a gene recombinant retrovirus.6. Transplantation of autogeneic tissue-engineered bone into rabbit segmental ulnar defects.Main results and conclusions:1. The results indicated that human marrow-derived MSCs can act as seeded cells for construction of cell-scaffold complex in bone tissue engineering. The MSCs can be isolated from human iliac marrow and changed to express alkaline phosphatase, collagen I, osteocalcin and regions of mineralization under under osteogenic differentiation conditions. There were 3.2 X 107~6.0X 107 mononuclear cells in 4 ml human bone marrow and 2.5 X 107~4.3 X 107 MSCs were obtained after 3 weeks of expanded-culture. It is feasible to apply MSCs construct cell-scaffold complex for clinical use.2. Cell density and medium exchanging had obvious affect on proliferation of MSCs. It is feasible that MSCs are cultured at a density of (1-3) X lO'/cm2 while medium should be changed after the first 48 hours during primary culture. In the expanded cultures for regenerated tissue-engineered bone, MSCs should be plated at 8X 103 /cm2. If MSCs has been enplated at 8 X 102 /cm2, cells colony can be formed, but many cells can't attach to the culture flask.3. MSCs can be differentiated into osteoblasts with some chemical drugs (Dexamethasone, P-Glycerphosphoric sodium, Vitamin C, etc) or special growth factor (recombination human bone morphogenetic protein-2 or bovine bone morphogenetic proteins). Chemical drugs were used to promote osteogenic differentiation, cells are not only expressed alkaline phosphatase and osteocalcin, but also produced regions of mineralization, moreover the results are credible and the cost of materials is cheap. So it can be regarded as standard technique of osteogenic differentiation. After differentiation, the proliferation of MSCs was degressive and the reduplication time changed from 36-40hours to 41-50 hours.4. Since the expression of EGFP or RFP showed no obvious effect on the proliferation and osteogenic differentiation of rabbit MSCs, it's a good method that MSCs labeled with fluorescent protein were used as seeded cells in tissue engineering. Compared with plasmid containing fluorescent protein, recombinant retrovirus is better to label MSCs.5. Fibrin gel was used in the new technique to construct cell-scaffold complex. Tissue fonnation was observed by fluorescence microscope and scanning electron microscopy. The number of seeded cells in the complex were analysed after they were digested from the scaffold. It has some advantages that the distribution of cells throughout the porous scaffold is homogeneous and the cell concentration is high. This technique is useful for bone tissue engineering.6. After MSCs were labeled with EGFP or 5-bromo-2-deoxyuridine (BrdU), autologous MSCs-Gelatin constructs and allogeneic MSCs-Gelatin constructs were implanted into rabbit subcutaneous tissue. Survival rate and migration of labeled MSCs in the constructions were analyzed at 3 days, 1 week, 3 weeks and 5 weeks after implantation. Cells expressing EGFP under fluorescence microscope or detected BrdU by immunohistochemical method in all the samples were observed during the initial five weeks after allogeneic or autologous MSCs-Gelatin constructs had been implanted. At early time points of 3 days and 1 week, inflammatory reaction was obviously observed, then inflammatory cells were diminution. By weeks 3 and 5 the labeled cells were extensive retained on the surface of gelatin sponge and gradually retained into host tissue. In a word, the rabbit allogeneic MSCs can survive at least five weeks after implantation into subcutaneous tissue and these cells are able to migrate obviously.7. The full-length human HIF-1 a cDNA was cloned into the retroviral vector containing internal ribosomal entry site (IRES) and enhanced green fluorescent protein (EGFP) by the method of gene engineering. Restriction endonuclease and PCR analyses confirmed that the recombinant retrovirus containing HIF-1 a gene was successfully constructed and it was important for therapeutic angiogenesis. The recombinant retrovirus could infect NIH3T3 cells and MSCs. Besides retroviral characteristic, the vector had IRES sequence and evaluation HIF-1 a expressed was able to observe EGFP expression with fluorescent microscope.8. The experiment repaired 1.5 cm rabbit segmental ulnar defects with different graft. The outcomes showed that transplantation of tissue-engineered bone was superior to only scaffold. Because new osseous tissue was more quantitative and regular and faster reformative in group of tissue-engineered bone than in group of scaffold. Nonunion had presented in group of no graft into bone defects.